First publ. in: Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral
Physiology ; 193 (2007), 9. - pp. 993-1000
DOI: 10.1007/s00359-007-0252-8
Reformation process of the neuronal template
for nestmate-recognition cues in the carpenter ant
Camponotus Xoridanus
Sara Diana Leonhardt · Andreas Simon Brandstaetter ·
Christoph Johannes Kleineidam
Abstract Ants use cuticular hydrocarbons (CHC-proWles)
as multicomponent recognition cues to identify colony members (nestmates). Recognition cues (label) are thought to be
perceived during ant–ant encounters and compared to a neuronal template that represents the colony label. Over time, the
CHC-proWle may change, and the template is adjusted
accordingly. A phenotype mismatch between label and template, as happens with CHC-proWles of foreign workers (nonnestmates), frequently leads to aggressive behavior. We
investigated the template reformation in workers of the carpenter ant Camponotus Xoridanus by masking their antennae
with postpharyngeal gland (PPG) extracts from nestmates or
non-nestmates. The behavioral response of manipulated
workers encountering unmanipulated workers was measured
independently after 2 and after 15 h. After 2 h of incubation,
workers treated with either of the two PPG-extracts showed
low aggression towards nestmates and high aggression
towards non-nestmates. In contrast, after 15 h of incubation,
workers treated with non-nestmate PPG-extract showed low
aggression towards both nestmates and non-nestmates. The
slow (>2 h) adjustment of the template indicates a reformation localized in the central nervous system rather than in
chemosensory neurons. In addition, our data show that template adjustment to a new CHC-proWle does not impair the
assessment of the old CHC-proWle as nestmate label.
Keywords Postpharyngeal gland · Cuticular
hydrocarbons · Chemical communication · Hymenoptera ·
Aggressive behavior
S. D. Leonhardt · A. S. Brandstaetter · C. J. Kleineidam (&)
Department of Sociobiology and Behavioral Physiology,
Biozentrum, University of Würzburg, Am Hubland,
97074 Würzburg, Germany
e-mail: [email protected]
Introduction
In social insects, colony survival depends on an elaborate
chemical system which ensures identiWcation of conspeciWcs, nestmates, and kin (Hölldobler and Wilson 1990;
Hölldobler 1995). Particularly, cuticular hydrocarbons
(CHCs) have been found to serve as chemical recognition
cues in ants (Howard 1993; Singer 1998; Lahav et al. 1999;
Lucas et al. 2005). The primary function of CHCs is to preserve insects from desiccation, cuticle abrasion, and infection, unless they became further involved in the social
recognition system of colony-building insects (Lockey
1988). Across colonies of the same species, CHC-proWles
consist of the same components, although in diVerent ratios
(Vander Meer and Morel 1998). Within a colony, CHC-proWles are not stable, but are inXuenced by environmental factors and vary with age, reproductive status, and/or caste
membership of their bearers (Morel et al. 1988; Wagner
et al. 1998; Nielsen et al. 1999; Heinze et al. 2002; Dietemann et al. 2003; D’Ettorre et al. 2004; Endler et al. 2004;
Buczkowski et al. 2005; Katzerke et al. 2006). The interindividual variation of CHC-proWles requires that the workers’ acceptance range for nestmate CHC-proWles is wide
enough to account for caste- or age diVerences. In contrast,
the temporal variation of the colony CHC-proWle implies
that ants permanently adjust and conWne their acceptance
range to allow a reliable discrimination from non-nestmates.
It is thought that information about the colony-speciWc
CHC-proWle is represented as a neuronal template in the
nervous system. During encounters with other individuals,
recognition cues (label) of the CHC-proWle are perceived
and compared to the template in a process described as phenotype matching (Crozier and Dix 1979; Lacy and Sherman
1983; Getz and Chapman 1987; Crozier and Pamilo 1996;
Hauber and Sherman 2001). A mismatch between label and
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template, as happens with CHC-proWles of non-nestmate
workers, generally results in aggressive behavior (Lacy and
Sherman 1983). The formation and adjustment of the template requires at least simple forms of learning (e.g. adaptation or habituation) and frequent interaction between
colony members to unify the colony label via trophallaxis
and allogrooming (Soroker et al. 1994; Dahbi et al. 1999).
The postpharyngeal glands (PPGs) play an important role
in this uniWcation, and their content shows a strong congruence to the cuticular CHC-proWle (Bagneres and Morgan
1991; Soroker et al. 1994). As a pool for inherent and exogenous hydrocarbons, the PPGs are used for distributing
CHCs such that all workers possess the same CHC-proWle
and share a so-called ‘Gestalt colony odor’. The misleading
term ‘Gestalt’ was introduced by Crozier and Dix (1979) in
the ‘Gestalt model’, probably to emphasize the complexity
and the emergent properties in the nestmate recognition
system. ‘Gestalt colony odor’ is still used and even led
some authors to term the postpharyngeal glands a ‘Gestalt
organ’ for nestmate recognition (Soroker et al. 1994;
Lenoir et al. 1999, 2001; Boulay et al. 2004; Akino and
Yamaoka 2005). It is important to note that the term
‘Gestalt’ in Psychology and in Neurobiology describes a
phenomenon of perception (von Ehrenfels 1890; Wertheimer 1925; Kandel and Wurtz 1990). Thus, a concept actually associated with the brain has been misleadingly
transferred to the chemical properties of the cuticle and the
postpharyngeal glands, confusing what is perceived and
what is detected. For the sake of consistent terminology, the
term ‘Gestalt’ may be used in the context of nestmate recognition in the same way as in Neurobiology and in Psychology, that is as a description of brain function.
It is not known, where the template is localized in the
nervous system. The template may be localized in the central nervous system (CNS). In case the template were localized in the CNS, aggression would be regulated by a
threshold of similarity between template and label (Crozier
and Dix 1979; Obin and Vander Meer 1989; Vander Meer
and Morel 1998). Alternatively, the adaptation of the
worker’s olfactory receptor neurons (ORNs) due to constant stimulation with its own CHC-proWle may function as
a template. The latter scenario was recently proposed by
Ozaki and colleagues who found that ORNs of one particular chemosensory sensillum were responsive only to CHC
extracts of non-nestmates, but were unresponsive to CHC
extracts of nestmates (Ozaki et al. 2005). Ozaki and colleagues suggested a peripheral recognition mechanism for
detection of colony-speciWc recognition cues.
Depending on the mechanism utilized for template reformation, the adjustment to a changing label is expected to
occur at diVerent time scales with peripheral adaptation
(ORN-adaptation) occurring faster than plasticity in the
central nervous system (CNS-plasticity).
In this study, we investigated the template reformation in
workers of the carpenter ant Camponotus Xoridanus following a manipulation of their sensory apparatus. The sensory experience of workers was manipulated by masking
their own CHC-proWle on the antenna with CHCs obtained
from PPGs of other (nestmate and non-nestmate) workers.
The behavioral response (aggressive behavior) of manipulated workers towards single unmanipulated nestmates or
non-nestmates was measured either after 2 h or after 15 h.
The change in behavior was used as a measure for the reformation process of the template, and the time of change for
reformation is discussed with respect to the two proposed
mechanisms, ORN-adaptation and CNS-plasticity.
Materials and methods
Animals
For behavioral experiments with manipulated workers we
used a laboratory colony of C. Xoridanus. The founding
queen of the colony (colony C316) had been collected by
Endler and Diederich at the Sugarloaf Shores, FL, USA, in
2003 and reared in an environmental chamber at 25°C and
50% relative humidity in a 12 h/12 h photoperiod. At the
time of the experiments, the colony had about 4,000 individuals and was provided with honeywater, dead cockroaches (Nauphoeta cinerea), and artiWcial diet (Bhatkar
and Whitcomb 1970), twice a week, and water ad libitum.
Only large workers (head with >3 mm) were used for
behavioral experiments.
PPG dissection and extract application
The postpharyngeal glands (PPGs) for manipulation of
other workers’ antennae were obtained from CO2 anesthetized small workers (head with <3 mm) by dissecting their
heads. The postpharyngeal glands were transferred to a
cleaned microscope slide and carefully squeezed with a
tweezers’ tip to open the glands and disperse the extract on
the slide. Immediately afterwards, an immobilized (on ice)
large worker was swayed headWrst above the microscope
slide with only its antennae touching the slide surface, so
that the PPG extract was evenly applied on its antennae.
This procedure lasted no longer than 30 s because the PPG
extract dried up rapidly. We used one gland per large
worker of C. Xoridanus. We established two groups of
manipulated workers according to diVerent PPG treatments
of their antennae. One group of workers was treated with
PPG extract from nestmates (colony C316), whereas the
other group received a treatment with PPG extract obtained
from workers of a foreign colony (non-nestmates; colony
C90), the founding queen of which had been collected by
995
Endler and Strehl on Orchid Island, FL, USA, in 2001. The
non-nestmate colony was similar in size and reared under
the same conditions as the nestmate colony.
Behavioral experiments with yoke-workers
Manipulated workers were restrained in a modiWed Petridish. We cut a slit into the rim of a Petri-dish that was wide
enough to let a large worker’s neck slip through (approx.
0.5 mm). Alike the yoke for oxen, the Petri-dish (yokedish) was overturned and put over the worker’s neck (similar to the immobilization device used by Lucas et al. 2005).
A piece of adhesive tape was placed above the worker’s
head to adjust its position, and the body posterior to the
neck was free to move inside the yoke-dish (Fig. 1a, c). The
yoke prevented the workers from cleaning their antennae.
However, the workers restrained in the yoke-dish (yokeworkers) were still mobile enough for behavioral assays.
The opening of the mandibles, movements of the antennae,
and movements of legs and gaster could be observed which
allowed us to describe behavioral responses of yoke-workers more accurately than it is possible with other devices
commonly used for behavioral experiments.
Yoke-workers were placed on a wooden board about
25 cm above a cleaned bench with the workers’ heads positioned at the rim of the board. Thus, the workers’ mandibles
and their manipulated antennae were located in midair.
We observed the behavioral response of yoke-workers in
controlled encounters with either nestmates (from colony
C316) or non-nestmates (from colony C90) after 2 h (61
individuals) or after 15 h (61 individuals). Five large workers were transferred to a wooden bridge of equal height to
the board with the yoke-workers (25 cm above the bench
top). The gap in-between the end of the bridge and the
board was small enough to allow antennation, but wide
enough to prevent the freely moving workers (henceforth
called encounter-workers) from crossing the gap or biting
the yoke-workers (Fig. 1c). As soon as one encounterworker from the bridge approached and antennated the
yoke-worker, the behavioral response of the yoke-worker
was observed during the Wrst 5 s of an encounter and scored
according to the following behavioral index:
–
–
–
–
–
0: no reaction
1: antennation, mandibles closed
2: mandibles slightly open
3: mandibles widely open
4: wide opening and immediate closing of mandibles
(“biting”)
This index had been established during several pre-testing
trials with untreated individuals. We recorded the highest
aggression response observed. In order to obtain a more
Fig. 1 Yoke-dish with a tethered worker of C. Xoridanus as used in
the behavioral experiments. a View from above showing the vertical
rim of the Petri dish (yoke-dish, white) which functions as a yoke for
the worker. b: Frontal view on the head of a yoke-worker, the neck is
Wxated by the gap of the yoke-dish. c: Interaction of a freely moving
worker (encounter-worker) with a yoke-worker. In the experiments,
the behavioral response of both, yoke- and encounter-worker, was recorded during an interaction
accurate description of the workers’ behavior, also intermediate scores were used when the behavioral response could
not be unambiguously assigned to an integer score.
In addition to the behavioral response of yoke-workers,
we further noted the behavior of encounter-workers to
count for any bias in the behavioral response of yoke-workers. Because encounter-workers initiated the behavioral
sequence of each encounter the behavioral response of
yoke-workers may be induced by the behavior shown by
the encounter-workers. We noted whether encounter-workers acted aggressively (open mandibles, attempts to attack)
or non-aggressively (closed mandibles, sometimes attempts
to perform trophallaxis).
Yoke-workers as well as encounter-workers were tested
individually and only once. The encounter-workers were
gently removed from the bridge after interaction with a
yoke-worker. The kind of PPG treatment the yoke-workers
had received was balanced on all days of experiments, and
the observer was blind to the PPG treatment. The nestmate
and non-nestmate encounter-workers (n-NM or NM
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encounter-workers) were presented to the yoke-workers
pseudo randomly.
4
NM encounter-workers
a
Statistical analysis
Results
n.s.
3
2
behavioral index of yoke-workers
The behavioral responses of yoke-workers was analyzed
separately for the groups tested after 2 h and the groups
tested after 15 h, using the Kruskal–Wallis ANOVA (KWtest) followed by multiple comparisons (mc) between
groups.
In order to analyze whether the behavior of encounterworkers correlates with the behavior of yoke-workers, we
calculated the Spearmans-Rank correlations (SR) for all
data pooled and for each group separately. If the behavioral
response of yoke-workers depends on the behavior of
encounter-workers we expect to Wnd the same diVerences in
behavior in both encounter-workers and yoke-workers.
Therefore, we tested the behavior of encounter-workers
between independent groups of nestmates and non-nestmates, using the Chi2-test (with Fishers exact p), and compared the diVerences to the diVerences found in the
behavior of yoke-workers.
All calculations were performed using the software Statistica 7.1 (StatSoft, Tulsa, OK, USA).
*
n.s.
(2 hours)
n-NM encounter-workers
1
0
4
* *
b
*
(15 hours)
3
2
1
0
NM
n-NM
NM
n-NM
extract applied on yoke-workers’ antennae
Workers restrained in the yoke-device quickly calmed down
within 3 to 5 min and adopted a posture with retreated
antennae. Only in response to disturbance, e.g. an encounter
with another worker, yoke-workers started moving their
antennae and mandibles. We recorded the behavioral
response of yoke-workers only for a short period, right after
onset of the interaction with encounter-workers, because,
later on, the yoke-workers tried to escape the yoke-dish by
pulling with their legs and bending their gaster. This behavioral pattern was similar irrespective of the time the yokeworkers were tested. The constancy in behavior indicates
that workers restrained in the yoke-device were in good
physical condition throughout the experiments.
Yoke-workers responded with low aggression (median
aggression score of 2 or less) when encountering a nestmate
(NM encounter-workers, Fig. 2a, b; left). The four groups
of yoke-workers tested after 2 h of incubation diVered signiWcantly in their behavioral response towards encounterworkers (Fig. 2a, KW-test: H(3;62) = 9.84; P < 0.05).
However, we found no diVerence between NM yoke-workers and n-NM yoke-workers, neither in encounters with
NM- nor in encounters with n-NM encounter-workers
(mc: P >> 0.05 for both pairs). Irrespective of treatment,
yoke-workers responded less aggressively towards NM
encounter-workers than towards n-NM encounter workers
Fig. 2 Behavioral responses of yoke-workers treated with nestmate
PPG extract (NM, light bars) and non-nestmate PPG extract (n-NM,
dark bars) towards nestmate (left side) and non-nestmate encounterworkers (right side). Yoke-workers were tested either after 2 h (a) or
15 h (b) of treatment with PPG extracts. Behavioral responses were
scored according to the behavioral index described in the Materials and
methods. Bars denote the median and whiskers the range of workers’
behavioral responses. Groups tested after 2 and 15 h were analyzed
separately using a Kruskal–Wallis-ANOVA with multiple comparisons. Asterisks indicate signiWcant diVerences at a level of P < 0.05, ns
not signiWcant
(pooled data, KW-test: H(1;62) = 8.80; P < 0.01, mc:
P < 0.01).
The four groups of yoke-workers tested after 15 h of
incubation also diVered signiWcantly in their behavioral
responses towards encounter-workers (Fig. 2b, KW-test:
H(3;60) = 13.11; P < 0.01). In encounters with NM
encounter-workers, yoke-workers showed low aggression,
irrespective of yoke-worker-treatment (Fig. 2b left, mc:
P >> 0.05). Interestingly, in encounters with n-NM encounter workers, n-NM yoke-workers now (after 15 h of treatment) showed signiWcantly lower aggression than NM
yoke-workers (Fig. 1b right, mc: P < 0.05). The NM yokeworkers were still aggressive towards n-NM encounterworkers. They were signiWcantly more aggressive towards
n-NM encounter-workers than both NM and n-NM
997
*
behavioral index of yoke-workers
4
3
2
1
0
hostile
friendly
behavior of encounter-workers
Fig. 3 The behavioral responses of encounter-workers (dichotomy
data, friendly or hostile) compared to the behavioral responses of yokeworkers. Bars denote the median and whiskers the range of workers’
behavioral responses. All 122 encounters were analyzed using a Spearmans rank correlation (R = 0.57; P < 0.05)
yoke-workers towards NM-encounter workers (Fig. 2b, mc:
P < 0.05 for both pairs).
The behavior shown by yoke-workers correlated with
the behavior shown by encounter-workers (Fig. 3). Yokeworkers had higher behavioral index scores when encounter-workers showed aggression (hostile) than when they
showed no aggression (friendly) (SR: R = 0.57; n = 122;
P < 0.05). A signiWcant correlation was found within Wve of
the eight groups, and in all four of the groups tested after
15 h of yoke-worker-treatment (SR: R = 0.49 to 0.63;
n = 13 to 17; P < 0.05 for all Wve groups, indicated by an
asterisk in Table 1). Note that only a behavioral index of 3
and more clearly indicates aggression; thus a correlation
between hostile encounter-workers and yoke-workers not
necessarily indicates aggressive behavior of yoke-workers.
The behavioral diVerence found in yoke-workers might
be inXuenced by the behavior of encounter-workers
(Table 1). When comparing encounters with yoke-workers
treated for 2 h, NM encounter-workers showed less often
aggressive behavior towards n-NM yoke-workers than nNM encounter-workers (Chi2 = 5.01, P < 0.05). Likewise,
NM encounter-workers showed less often aggressive
behavior towards NM yoke-workers than n-NM encounterworkers (Chi2 = 7.80, P < 0.01).
When comparing encounters with yoke-workers
treated for 15 h, aggression occurred less often in NM
encounter-workers (in about 50% of the cases) than in nNM encounter-workers (in about 80% of the cases). We
found no inXuence of the yoke-worker-treatment, neither
on the behavioral response of NM encounter-workers
(Chi2 = 0.03, P = 0.58) nor on the behavioral response of
n-NM encounter-workers (Chi2 = 0.04, P = 0.44). Note
that, in contrast, the behavioral response of yoke-workers in such encounters diVered signiWcantly (Fig. 2b
right).
For all pairs of independent groups, the encounter-workers’ behavior corresponded to the behavior observed in
yoke-workers, except for one pair: After a treatment of
15 h, n-NM yoke-workers showed low aggression scores
towards n-NM encounter-workers but, in reverse, n-NM
encounter-workers showed aggressive behavior towards
NM- and n-NM yoke-workers equally often.
Discussion
Our study shows that the sensory experience of immobilized workers of the carpenter ant Camponotus Xoridanus
can be manipulated with non-nestmate PPG extracts and
that the manipulation later leads to a change in behavior.
Tethered workers (yoke-workers) treated with non-nestmate PPG extract showed a reduction in aggressive behavior towards freely moving non-nestmates, after 15 h of
antennal treatment. However, after 2 h of treatment, they
showed a similar behavioral response towards encounterworkers than yoke-workers treated with nestmate PPG
extract.
Table 1 Behavioral responses of encounter-workers towards yoke-workers
NM encounter-worker
NM yoke-worker
2h
n-NM yoke-worker
NM yoke-worker
n-NM yoke-worker
Friendly (n)
7
9
1
2
Hostile (n)
7
8
16
12
14
17*
17
14
9
7
2
4
Total (n)
15 h
n-NM encounter-worker
Friendly (n)
Hostile (n)
Total (n)
8
7
11
12
17*
14*
13*
16*
* Denote signiWcant correlation between encounter-workers behavior and yoke-workers behavior (Spearmans-Rank correlation; R = 0.49 to 0.63;
P < 0.05)
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We used immobilized yoke-workers to prevent any
interaction with other workers prior to behavioral testing
and to provoke a change in the neuronal template only due
to constant stimulation of the ants’ antennae. Yoke-workers
treated with PPG extracts displayed a low level of aggression towards encountering nestmates (median aggression
scores of ·2, only scores ¸3 clearly indicate aggression).
Similarly low-aggression scores were measured with
untreated yoke-workers encountering nestmates, in the pilot
experiments that we used to establish the aggression index
(data not shown). These results and the Wnding of speciWc
aggression of yoke-workers towards non-nestmates indicate that the antennal treatment itself has little impact on
the general discrimination ability of workers.
Response to non-nestmates
We found that yoke-workers were highly aggressive
towards non-nestmates, after 2 h following the treatment
with either PPG extract. After 15 h, however, yoke-workers
showed a diVerent behavioral response towards non-nestmates. Their behavioral response depended on the kind of
treatment they had received: Yoke-workers treated with
non-nestmate PPG extracts showed a lower level of aggression than yoke-workers treated with nestmate PPG extract.
Is this change in behavior of yoke-workers treated with
non-nestmate PPG extract caused by the prolonged antennal treatment or is it induced by a change in behavior of
encounter-workers? We are conWdent that the observed
change in aggressive behavior of yoke-workers was provoked by the antennal treatment and not by a change in
behavior of encounter-workers because non-nestmate
encounter-workers more often showed aggressive behavior
than nestmate encounter-workers, irrespective of the treatment the yoke-workers received. In particular, non-nestmate encounter-workers often showed aggressive behavior
towards yoke-workers treated with non-nestmate PPG
extract for 15 h although these yoke-workers showed less
often aggressive behavior than yoke-workers treated with
nestmate PPG extract. This result seemingly contradicts the
correlation found between the behavior of non-nestmate
encounter-workers and non-nestmate yoke-workers after
15 h treatment (Fig. 2, Table 1, bottom right). Since only
few of the yoke-workers in this group had a behavioral
index of 3 or more (clearly aggressive behavior, 3 out of
16), the correlation probably describes a stronger but nonaggressive behavioral reaction of yoke-workers towards
aggressive non-nestmate encounter-workers.
In addition to providing evidence for the importance of
chemical cues, this result indicates that visual cues at most
play a minor role in the nestmate recognition process of
yoke-workers. However, it might well be that free running
workers may use both, chemical and visual cues for nestmate
recognition because perception of visual stimuli often
requires self-generated optical Xow (Hori et al. 2006).
Our results indicate a label-template matching of the
non-nestmate label due to a reformation of the template in
yoke-workers treated with n-NM PPG extract. Reformation
of the template may start right after manipulation but it
takes at least 2 h for functional adjustment of the template.
Given that ant CHC-proWles are not stable but inXuenced
by environmental factors and that they vary with age,
reproductive status, and caste membership of their bearers,
it is expected that the neuronal template used for phenotype
matching expresses plasticity to allow for a rapid behavioral change (Morel et al. 1988; Wagner et al. 1998; Nielsen et al. 1999; Heinze et al. 2002; Dietemann et al. 2003;
D’Ettorre et al. 2004; Endler et al. 2004; Buczkowski et al.
2005; Katzerke et al. 2006). This template plasticity
appears to primarily result in an extension of behavioral tolerance as has been demonstrated for several ant species
where individuals of one species expressed a broader
acceptance range towards individuals of a diVerent species
when they had been reared in mixed species groups (Errard
et al. 2006). Errard et al. showed that greater dissimilarity
in the CHC-proWles of workers in mixed species groups
resulted in a broader acceptance range based on a broader
template rather than on multiple templates.
Response to nestmates
Yoke-workers treated with either PPG extract showed no
diVerent behavioral response towards nestmates, neither
after 2 h nor after 15 h of treatment. Thus, the acceptance
range of manipulated workers is broadened, after 15 h of
non-nestmate treatment, with phenotype matching to the
nestmate label as well as to the non-nestmate label. It
remains to be examined whether, on a longer time scale,
template reformation will lead to further adjustment by
constriction of the template’s acceptance range (Wne-tuned
template) to the sensory experience, which, in our experiment, is represented by the non-nestmate label. In honeybees, the corporate fostering of workers from diVerent,
unrelated colonies also leads to a change in template (Harano and Sasaki 2006). Harano and Sasaki also found that it
takes more than a day of exposure to non-nestmates before
workers start to respond aggressively towards their former
nestmates indicating a lagged Wne-tuning.
Temporal dynamic of template reformation
It is expected that the reformation of the template is adapted
to the temporal structure of naturally occurring changes in
the CHC-proWles. In honeybees, the template reformation
occurs within 25 min of exposure (Harano and Sasaki
2006), whereas in our study it took more than 2 h. This is a
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much faster adjustment. Possibly, the Xower-scent loaded
nectar collected by honeybees cause faster changes in the
CHC-proWle of the colony than the food collected by C.
Xoridanus. Unlike to our experiment, in other experiments
investigating the template reformation of ants and bees,
individuals were allowed to mutually and frequently interact with non-nestmates. They were able to exchange CHCs
(Vienne et al. 1995) and to frequently resample the CHCproWles of nearby non-nestmate workers. The inXuence of
repeated interaction and sampling of the non-nestmate
workers’ CHC-proWle may inXuence the process of template reformation which remains to be investigated.
The transient time for a change in the colony CHC-proWle
results in a larger variation of worker CHC-proWles within a
colony. To account for such variation, template reformation
is expected to result in a broadened acceptance range of
workers which is supported by our and others’ results. The
template reformation process seems to consist of this initial
phase, and a delayed phase in which the template is Wnetuned. These two phases require a neuronal plasticity which
hardly can be implemented at the level of the olfactory
receptor neurons, even if many of them are assembled as a
functional unit in one sensillum. Recently, Ozaki et al. proposed that the sensory response at a chemosensory sensillum
may function as template (Ozaki et al. 2005). The authors
suggested a speciWc adaptation of the response of multiple
receptor neurons to nestmate CHC-proWles as a possible
mechanism. Even if the proposed mechanism of a complex
adaptation to ratios of multiple components exists, it cannot
explain the two phases of template reformation process
described above yet. For insect olfactory receptor neurons,
several diVerent adaptation mechanisms were described
(Kurahashi and Menini 1997; Redkozubov 2000; Dolzer
et al. 2003). However, all of them are acting on a shorter
time scale as the behavioral change described in this study
(>2 h) and, presumably, even on a shorter time frame as the
faster template reformation process (25 min) found in honeybees (Vareschi 1971; Bittel and Martin 1974; Kaissling
et al. 1987; Harano and Sasaki 2006). On a longer time
scale, neuromodulators (e.g. octopamin and serotonin) may
modify the sensory response as has been shown for the circadian change of olfactory receptor neuron sensitivity (Pophof 2000; Dolzer et al. 2001; Flecke et al. 2006).
Neuromodulation acting on olfactory receptor neurons represents a global adjustment of sensitivity. Nevertheless, it is
also unlikely to be a mechanism for such speciWc template
formation as described in ants and honeybees. The slow reformation process of the template and its Wne-tuned adjustment suggest CNS-plasticity rather than ORN-adaptation as
a mechanism for template formation. However, further
physiological studies on ORNs (EAG or single unit recordings) are necessary to investigate possible inXuences of sensory adaptation on nestmate recognition.
Our Wnding supports the hypothesis that plasticity of the
central nervous system adjusts for changes in the CHC-proWle of the colony, with aggression being regulated by a
threshold of similarity between template and label (Crozier
and Dix 1979; Obin and Vander Meer 1989; Vander Meer
and Morel 1998).
Our results show that direct experimental manipulation
of the workers’ sensory experience can change the phenotype matching of their template. This technique can be used
as a tool to study the template reformation process not only
in behavioral experiments but also in physiological experiments investigating the representation of CHC-proWles in
the ant’s brain.
Acknowledgments We thank Annett Endler for collecting and taking
care of the ant colonies that we used as well as for constructive comments
on the experimental design as well as on this manuscript. We thank Linda S. Kuebler and Christina Kelber for helping with the experiments and
Annette Laudahn for advice in rearing the colony. This study was funded
by DFG, SFB 554/A6. The experiments comply with the “Principles of
animal care”, publication No. 86-23, revised 1985 of the National Institute of Health, and also with the current laws of Germany.
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